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The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases. Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

Carotid bodies (CBs) are chemoreceptors that monitor and register changes in the blood, including the levels of oxygen, carbon dioxide, and pH, and regulate breathing. Enhanced activity of CBs was shown to correlate with a significant elevation in the blood pressure of patients with hypertension. CB removal or denervation were previously shown to reduce hypertension. Here we demonstrate the feasibility of a dual-mode ultrasound array (DMUA) system to safely ablate the CB in vivo in a spontaneously hypertensive rat (SHR) model of hypertension. DMUA imaging was used for guiding and monitoring focused ultrasound (FUS) energy delivered to the target region. In particular, 3D imaging was used to identify the carotid bifurcation for targeting the CBs. Intermittent, high frame rate imaging during image-guided FUS (IgFUS) delivery was used for monitoring the lesion formation. DMUA imaging provided feedback for closed-loop control (CLC) of the lesion formation process to avoid overexposure. The procedure was tolerated well in over 100 SHR and normotensive rats that received unilateral and bilateral treatments. The measured mean arterial pressure (MAP) exhibited measurable deviation from baseline 2–4 weeks post IgFUS treatment. The results suggest that the direct unilateral FUS treatment of the CB might be sufficient to reduce the blood pressure in hypertensive rats and justify further investigation in large animals and eventually in human patients.

The GM2-gangliosidoses are neurological diseases causing premature death, thus developing effective treatment protocols is urgent. GM2-gangliosidoses result from deficiency of a lysosomal enzyme β-hexosaminidase (Hex) and subsequent accumulation of GM2 gangliosides. Genetic changes in HEXA, encoding the Hex α subunit, or HEXB, encoding the Hex β subunit, causes Tay–Sachs disease and Sandhoff disease, respectively. Previous studies have showed that a modified human Hex µ subunit (HEXM) can treat both Tay–Sachs and Sandhoff diseases by forming a homodimer to degrade GM2 gangliosides. To this end, we applied this HEXM subunit in our PS813 gene editing system to treat neonatal Sandhoff mice. Through AAV delivery of the CRISPR system, a promoterless HEXM cDNA will be integrated into the albumin safe harbor locus, and lysosomal enzyme will be expressed and secreted from edited hepatocytes. 4 months after the i.v. of AAV vectors, plasma MUGS and MUG activities reached up to 144- and 17-fold of wild-type levels (n = 10, p

Deficiencies in the lysosomal hydrolase β-galactosidase (β-gal) lead to two distinct diseases: the skeletal disease Morquio syndrome type B, and the neurodegenerative disease GM1-gangliosidosis. Utilizing CRISPR-Cas9 genome editing, the mouse β-gal encoding gene, Glb1, was targeted to generate both models of β-gal deficiency in a single experiment. For Morquio syndrome type B, the common human missense mutation W273L (position 274 in mice) was introduced into the Glb1 gene (Glb1W274L), while for GM1-gangliosidosis, a 20 bp mutation was generated to remove the catalytic nucleophile of β-gal (β-gal−/−). Glb1W274L mice showed a significant reduction in β-gal enzyme activity (8.4–13.3% of wildtype), but displayed no marked phenotype after one year. In contrast, β-gal−/− mice were devoid of β-gal enzyme activity (≤1% of wildtype), resulting in ganglioside accumulation and severe cellular vacuolation throughout the central nervous system (CNS). β-gal−/− mice also displayed severe neuromotor and neurocognitive dysfunction, and as the disease progressed, the mice became emaciated and succumbed to the disease by 10 months of age. Overall, in addition to generating a novel murine model that phenotypically resembles GM1-gangliosidosis, the first model of β-galactosidase deficiency with residual enzyme activity has been developed.

In the present study, we aimed to assess the toxicity of artemisinin on the haematological system and its effect on the productive performance of broiler chickens. Eighteen-day-old chickens were randomly divided into four groups of 30 chickens (three replicates of 10 broilers/group): control group and three experimental groups: ART5 - diet with 5 ppm of artemisinin; ART50 - diet with 50 ppm of artemisinin; and ART500 - diet with 500 ppm of artemisinin. Artemisinin enhanced the productive performances of broiler chickens at the lowest concentration (5 ppm), but at the highest concentration (500 ppm), it negatively affected weight gain and the feed conversion ratio. The performance characteristics of the chickens whose diets were supplemented with 50 ppm artemisinin were similar to those of the control group. Additionally, 5 ppm artemisinin in feed did not significantly affect the haematological parameters of the chickens, but 50 and 500 ppm artemisinin induced a gradual decline of the total leukocytes, lymphopenia, monocytosis, and eosinopenia, and the highest concentration caused anaemia. Artemisinin at a low concentration could be used as a feed additive in the poultry industry to improve organic broiler production performance with no serious side effects.

Tobacco is a complex chemical mixture, containing many toxicants and carcinogens. Most rodent risk assessment studies have focused on single chemicals or the complicated mixtures of tobacco smoke or its fractions. There are few studies evaluating how specific chemicals interact with one another to form the potent carcinogenic mixture of tobacco smoke. We hypothesized that tobacco smoke aldehydes like formaldehyde and acetaldehyde could enhance the carcinogenic properties of the tobacco-specific nitrosamines, N’-nitrosonornicotine (NNN) and 4-methylnitrosamine-1-(3-pyridyl)-1-butanone (NNK), through a variety of mechanisms. This hypothesis was tested in two established rodent tumor models, the NNN-induced rat esophageal tumor model and the NNK-induced A/J mouse lung tumor model. In the first model, rats were exposed to 0, 4, or 8 ppm NNN in the drinking water in the presence or absence of 3000 ppm acetaldehyde for up to 100 weeks. The number of esophageal papillomas per rat was doubled in animals receiving both acetaldehyde and 8 ppm NNN (0.5 versus 1.15 tumors/rat, respectively). Acetaldehyde alone did not cause esophageal tumors. DNA adduct levels were not affected by the combination of the two chemicals. In the second model, A/J mice were exposed to NNK (i.p, 0, 2.5, or 7.5 μmol in saline) in the presence or absence of acetaldehyde (0 or 360 ppmv) or formaldehyde (0 or 15 ppmv) for 3 hours in a nose-only inhalation chamber. Lung tumors were counted 16 weeks later. Neither aldehyde by itself induced lung tumors. However, mice receiving both NNK and acetaldehyde or formaldehyde had an increased number of adenomas with dysplasia or progression than those receiving only NNK, suggesting that aldehydes may increase dysplasia in tumors initiated by NNK. As in the rat study, DNA adduct levels were not affected by the coexposure. In a separate experiment, we tested the hypothesis that the elevated levels of carbon dioxide in tobacco smoke could affect the carcinogenic properties of NNK in the A/J mouse; tobacco smoke contains 12.5% carbon dioxide. Mice received a 3 h nose-only carbon dioxide (0, 5, 10, or 15%) coexposure of mice receiving NNK (i.p. in saline, 0, 2.5, or 7.5 μmol). The coexposure to carbon dioxide more than doubled the number of lung adenomas induced by 2.5 μmol NNK, with the maximal effect observed with 10% carbon dioxide (0%: 1.8 ± 1.9; 5%: 3.9 ± 2.8; 10%: 7.1 ± 3.5; 15%; 5.5 ± 2.9 lung adenomas/mouse). Lung adenomas were also significantly increased in mice receiving 7.5 μmol NNK, although to a lesser extent (0%: 11 ± 6.2; 5%: 13 ± 7.9; 10%: 18 ± 6.0; 15%; 12 ± 5.3 lung adenomas/mouse). This additive and synergistic effect of carbon dioxide was highly significant (p value = 1.0 × 10-14). The mechanism of this interaction is under investigation. Collectively, these studies support the hypothesis that the aldehydes and carbon dioxide present in the tobacco mixture interact to enhance the carcinogenic potency of the tobacco specific nitrosamines. (Funded by CA-184987.) Citation Format: Lisa A. Peterson, Marissa K. Oram, Donna E. Seabloom, William E. Smith, Alessia Stornetta, Karin R. Vevang, Monica Flavin, Alexandru F. Tabaran, Ingrid Cornax, M. Gerard O’Sullivan, Pramod Upadhyaya, Lin Zhang, Stephen S. Hecht, Silvia Balbo, Timothy S. Wiedmann. Interactions between tobacco smoke chemicals in rodent tumor models [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A19.

Background Current animal models of glioma are limited to small animal models, which are less predictive of treatment of human disease. Canines often develop gliomas de novo, but the natural history of the disease is not well described. Objective We provide data for naturally occurring canine gliomas; evaluating medical and surgical therapies. Methods We reviewed medical records of pet dogs with a presumptive diagnosis of glioma from MRI imaging that underwent surgery as part of the Canine Brain Tumor Clinical Trials Program. Breed, age, sex, median progression-free, and overall survival times and cause of death were recorded for multivariate analysis. Results Ninety five dogs (56 male; mean age = 8.3 years) were included, but nine were excluded as final pathology was non-neoplastic. Gross total resection was reported in 81 cases based on postoperative MRI. Seventy had high-grade tumors (grade III or IV). Eighty three dogs presented with seizures, being the most common presenting clinical sign. Median survival after surgery was 723 days (95% CI 343-1103) for grade II tumors, 301 days (197-404) for grade III and 200 days (126-274) for grade IV (p = .009 Kaplan-Meier survival analysis; Log Rank test). Age (cox regression, p = .14) or sex (Kaplan-Meier test, p = .22) did not predict survival. Conclusions This study establishes normative data for a model exploiting dogs with naturally occurring glioma, which can be used to test novel therapies prior to translation to human trials. Further work will focus on the effects of different therapies, including chemotherapy, radiation therapy, and immunotherapy.

Osteoarthritis (OA) represents a public health challenge since the pathogenic treatment, able to induce cartilage regeneration, still remains unknown. Ageing of the population and increasing OA prevalence have led to a lot of research, aiming to identify treatments acting on chondrocytes that play a determinant role in cartilage degeneration÷regeneration balance. Pulsed shortwave therapy (with the classical application form - Diapulse) is a physiotherapy method with anabolic effects demonstrated on nervous, conjunctive and vascular tissues, but its effects on OA cartilage are not known.Our aim was to demonstrate the effects of Diapulse on the cartilage in experimental induced OA.Experimental OA was induced in 10 mature female rabbits by anterior cruciate ligament transection (ACLT). Ten weeks after ACLT, rabbits were randomized in a treatment group and a control group. Treatment group was exposed to Diapulse at a frequency of 27.12 MHz, pulse length of 65 μs, pulse frequency of 300 pulses÷s (300 Hz) for 10 minutes÷day. Control group was exposed to sham therapy. After treatment, rabbits were sacrificed and the cartilage was evaluated by histopathological examinations with Hematoxylin-Eosin (HE) staining and transmission electron microscopy (TEM).OA characteristic changes were found in both groups. In the treatment group, we found that Diapulse influenced the degenerative process in the OA cartilage by improving the chondrocyte viability and the capacity to maintain cellular matrix integrity and structure.Diapulse can be considered a disease modifying therapeutic procedure and could be a reliable option for treatment of OA patients.

The aim was to reveal the prevalence of post-mortem lesions in two large-scale bovine slaughterhouses found in Romania so as to point out the potential hazards for human health. The proper examination of animal carcasses and organs that are destined for human consumption is essential in order to ensure food safety but not all the time properly made due to lack of time during the process of slaughter. The results showed that the most frequent lesions found within the examined organs were caused by parasitic infestation (Echinococcus granulosus; Dyctiocaulus viviparus) and the highest prevalence was found in the lungs. The pathology of lesions found in bovine organs is very variable in the slaughterhouse, being able to report strange aspects such an ectopic liver in the lung parenchyma. Measures should be taken in the farms due to this high prevalence of parasites which causes both health problems as well as economical losses.

Anticoagulant rodenticides are widely used pesticides globally. They act by inhibiting vitamin K epoxide reductase in the liver, decreasing the synthesis of clotting factors II, VII, IX, and X. Sixty six cases of anticoagulant rodenticide toxicosis were found during a retrospective study conducted at the Veterinary Hospital and the Pathology Department of the Faculty of Veterinary Medicine Cluj-Napoca, in a period between January 2010 and June 2015.The inclusion criteria were: clinical signs of exposure, coagulation testing result and typical pathological findings characteristic for bleeding syndrome. History, clinical and pathological data were collected from the medical records of the dogs presented to our units. In the present study, 66 cases of anticoagulant toxicosis in dogs were described, the animals ranging in age from 2 months to 12 years. 31 dogs were male and 35 female. Two cases followed exposure to brodifacoum, 4 cases exposure to warfarin and in the other cases the type of anticoagulant was unknown. The most common clinical signs were dyspnea, lethargy, weakness, anemia/palor, melena, spontaneous subcutaneous and muscular hematoma, sublingual hematoma and ecchymoses. The most important lesions were represented by haemothorax, thymic hematoma in juvenile dogs, subcutaneous and muscular hematomas, hemoperitoneum, severe anemia, and ecchymotic hemorrhages in different tissues. In cases were coagulation testing was performed, prolonged partial thromboplastin time, prothrombin time and coagulation time were recorded. Anticoagulant rodenticide toxicosis is frequently found in our region, the most important diagnostic tools being coagulation testing and toxicological exam.

Primary spindle cell neoplasms of the gastrointestinal system show considerable morphologic overlaps. Proper histological classification of these neoplasms using only the classical hematoxylin-eosin (HE) stained slides is often difficult and frequently a source of diagnosis error. Thus, the immunohistochemistry is an essential tool in the proper diagnosis and classification of such tumors.In this study we tested and standardised an immunohistochemical protocol used for the differentiation of primary intestinal sarcomas (nonangiogenic, nonlymphogenic intestinal sarcomas) in dogs. The paper also aimed to critically review the state of art in immunohistochemistry of intestinal spindle cell neoplasms currently used in dogs.Seven cases of primary intestinal spindle cell neoplasms were diagnosed and classified using a 6 antibody panel immunohistochemistry. Indirect immunohistochemistry staining for S100 protein, desmin, I±-SMA, CD34, c-Kit and GFAP was conducted using a Leica Bond-Max auto-immunostainer.Based on the immunoreactivity, five stromal tumours were classified as leiomyosarcomas, one fibrosarcoma and one an undifferentiated sarcoma.In most cases of intestinal sarcomas, the proper diagnosis requests the use of immunohistochemistry. The retrospective analysis of these tumours proved that at least in one case, the HE stain was not sufficient for the proper diagnosis and classification of the tumour.

Aim: The aims of this study are the development of a contrast enhanced ultrasonography (CEUS) protocol for rats’ evaluation and the assessment of the potential benefits of CEUS in Walker 256 tumor rats. Material and method: In the study were used 36 albino Wistar rats grafted subcutaneously with Walker 256 tumor. The implementation of the ultrasound (US) guided injection technique (30 subjects – group A) was performed between 4 to 8 weeks after implantation. The contrast agent (CA) Sono Vue (Bracco) – was administered either into the lateral vein of the tail or directly into the heart under US guidance. The US validation, focusing on CEUS (6 subjects – group B) was realized at 4 to 6 weeks after implantation. The US procedures aimed to obtain morphological (2D), vascular (color Doppler and pulsed Doppler) and angiospecific functional data (CEUS). The Vevo 2100 equipment was used for US and Time Intensity Curves (TIC) were analyzed with Sonoliver (TomTec). The tumor specimens which were resected after the last study underwent a pathology exam. Results: A number of 23 successfully CEUS explorations were performed in 17 subjects (11 in group A and 6 in group B). Nineteen rats could not be evaluated (in 8 cases the tumor was not viable; 4 subjects died during CA administration; in 4 cases the administration line could not be obtained). In group B, CEUS was performed in 6 subjects at 4 weeks after implantation and in 5 subjects at 6 weeks. The statistical analysis of the TIC parameters identified significant differences between the Time to Peak, mean Transit Time and Rise Time parameters of the muscles and those of the tumor. Conclusions: CEUS was easily implemented on the studied tumor model and is adequate for the evaluation of tumor vascularity. US guided intracardiac administration of the CA is an easy and reproducible procedure. If the examination is performed at defined time intervals it detects the alterations within the tumor circulatory bed.